Streptomycin and the related aminoglycoside antibiotics, are currently of great clinical usefulness. Despite intense interest and study over the last forty years, the mode of action of streptomycin remains controversial. Historically, research has focused on two major aspects of streptomycin action: interactions with the bacterial ribosome, and uptake (membrane transport) into the bacterial cell. Major questions remain in both areas and, in addition, the interrelationship between membrane tansport and ribosomal binding is largely unexplored. The overall goal of this study, therefore, is to delineate the in vivo interaction of streptomycin with membrane-bound polysomes and correlate this effect with streptomycin uptake and killing. Specific experiments will be carried out to determine the following: (1) the kinetics of streptomycin binding to ribosomes in vivo as compared with the kinetics of 3H-streptomycin uptake under the same conditions; ribosomes will be separated into membrane-bound and free pools, (2) the effects on streptomycin ribosomal binding in vivo (membrane bound vs. free) of antibiotics which either promote (puromycin, penicillin) or suppress (chloramphenicol) streptomycin uptake and killing, (3) the effects on both streptomycin uptake and membrane attachment of polysomes of specific mutations in ribosomal RNA, (4) the effect of mutation in ribosomal protein S12 on attachment of polyribosomes to the membrane, (5) the effects on membrane-binding of polysomes of agents which increase or collapse the membrane potential (e.g. ionophores), and (6) the effects on streptomycin uptake and membrane-bound polysomes of augmented production of a secreted protein (beta-lactamase). 3H-streptomycin uptake will be determined by filtration assays. Membrane potential will be determined by measuring the steady-state distribution of the lipophilic cation 3H-tetraphenylphosphonium. Membrane-bound and free polysomes will be obtained by centrifugation in a discontinuous sucrose gradient and ribosomal binding in vivo will be determined by alterations in electrophoretic mobility of polysomes in composite gels. Organisms with mutant plasmids coding for altered rRNA have been constructed by site-directed mutagenesis.